Modular transfection systems

ABSTRACT

The present invention relates to a method for transfection of cells using at least one protein capable of forming nucleoprotein filaments, wherein the protein is initially modified with at least one functional component which influences one or more steps of the transfection, the nucleic acid to be transfected is then loaded with the modified protein, whereby the nucleic acid and the protein form a filament-like complex, and this complex is finally added to the cells to be transfected. The invention further relates to a transfection agent consisting of nucleoprotein filaments (NPF), with at least one nucleoprotein filament-forming protein being modified with at least one functional component for the transfection. Furthermore, the present invention relates to the use of the transfection agent according to the invention for producing a drug for gene therapeutic treatment of humans and animals. The present inventions also includes corresponding pharmaceutical preparations, especially for use in gene therapy as well as the use of such transfection agents as component in kits.

This application is a continuation of application of U.S. patentapplication Ser. No. 10/466,368, filed Aug. 13, 2003, which isincorporated herein by reference in its entirety and which is thenational stage entry of PCT/DE02/00060, filed Jan. 10, 2002 designatingthe United States and, which in turn claims priority from Germanapplication no. 101 00 996.8, filed Jan. 10, 2001, both of which arealso incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to the field of non-viral transfection ofnucleic acids. The term transfection generally means the introduction offoreign substances into cells. The present invention relates to a methodfor the transfection of cells with the help of at least one proteinwhich is capable of forming nucleoprotein filaments. The invention alsorelates to a transfection agent which contains a nucleoprotein filament(NPF) which is formed from at least one nucleic acid to be transfectedand at least one protein which is capable of forming nucleoproteinfilaments. In addition, the present invention relates to the use of thetransfection agent according to the invention, a correspondingpharmaceutical formulation, in particular for use in gene therapy, a kitfor the transfection of cells with nucleic acids and particular methodswhich use the transfection agent according to the invention.

STATE OF THE ART

The known non-viral transfection agents are subject to a series ofrestrictions. In non-viral transfection of nucleic acids, both the sizeof the globular nucleic acid complexes, which are often unilaterallycharged, and the lack of controllability of one or several transfectionsteps are usually a major problem. One cause of the latter is theinadequate ability to penetrate the external cell membrane or themembranes of internal compartments, inadequate protection from enzymaticdegradation, low bioavailability and biological effects of inadequatecontrollability caused by non-biological molecules in the cell. Nucleicacids mostly contain highly expanded steric structures, are easilydegradable by enzymes, and their unilateral charge excess causes readyassociation with basic cell structures. The associated inadequateability to pass into the nucleus has the result that the current nucleicacid transfection technologies almost exclusively employ cancer-liketransformed cells, such as cell lines, as the nuclear membrane in thesecells is temporarily disintegrated during cell division and entry intothe nucleus is possible. However, transformed cells are not comparableto the original physiological state of primary cells, so thatconclusions about the behavior of primary transfected cells cannot bereliably based on studies using transformed cells.

A series of transfection agents is already known, almost all of whichform globular complexes with nucleic acids by electrostatic interaction,often with a diameter of more than 50 nm (Tang and Szoka, 1997). Thesecomplexes mostly associate at the cell surface with a large excess ofcharge and are taken up by endocytosis. They leave the endosomes, eitherby buffering the acidification of the endosomes until these burst (e.g.with polyethylenimine, starburst dendrimers (Kukowska-Latello et al.1996) or addition of chloroquine), or by the action of membrane-activegroups, lipids or lipophilic peptides. However, the complexes can onlyreach the cell nucleus during the next cell division to show there thedesired effects after the nucleic acid has been released.

The problem of the division-dependent nuclear import can be avoided bythe use of NLS-peptides (NLS=nuclear localization signal), particularlywhen attention is paid to the problems of signal masking andnon-specific protein binding (WO 00/40742, Amaxa).

U.S. Pat. No. 5,468,629 describes the use of the RecA protein and thepossible use of other proteins with functional homology to RecA in thetransfection of cells with ssDNA (single-strand DNA). The protein RecAsupports, evidently by catalysis, the process of homologousrecombination of the transported ssDNA with the cell DNA by influencingstrand pairing and the subsequent strand exchange. The complexes usedhere contain ssDNA of maximally 700 nucleotides as well as RecA proteinand are described as “RecA-coated” complexes. Such ssDNA-proteincomplexes are formed from DNA in the presence of RecA and ATP-γ-S, withthe formation of stable helical presynaptic filaments. These complexesare used with little success, for example, for the transfection of celllines, i.e. for actively dividing cells, in particular transformedcells.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide a method and atransfection agent for the transfection of nucleic acids of any sortinto cells of any sort, which permit improved uptake in cells and whichat the same time allows the control of the transfection process. Thetransfection agent for this purpose should both be adequately stableduring the transfection process and also guarantee adequate release ofthe nucleic acids to be transfected in the target cell compartment.

This object is solved according to the invention with a method of thetype mentioned in the introduction, wherein the protein is initiallymodified with at least one functional component which influences one ormore steps of the transfection. The nucleic acid to be transfected isthen loaded with the modified protein, the nucleic acid and the proteinforming a filament-shaped complex, and this complex is finally added tothe cells to be transfected.

In addition, this object is solved by a transfection agent of the typementioned in the introduction, wherein the protein that is capable offorming nucleoprotein filaments is modified with at least one componentinfluencing the transfection.

As a result of the modification of one or several proteins which formnucleoprotein filaments (NPF) with one or several of the same ordifferent additional functional components, individual steps of thecomplex transfection process can be controlled in a particularlyadvantageous manner which is specific for the nucleic acid to betransfected and for the target cell. In addition, the method andtransfection agent according to the invention permit a clear increase intransfection efficiency in comparison with known methods.

According to the invention, the NPF-forming proteins are used in anadvantageous manner, particularly as modular carrier system forfunctional groups, which goes beyond pure protection of DNA. The complexexhibits very low extension in space in two respects: it isfilamentous—not globular—and it contains only one nucleic acid moleculeper filament. Because of the assembly which leads to a stoichiometricratio of a few nucleotides per carrier protein, an extremely highdensity of functional signals for transfection is possible, much morethan with globular complexes. The signal density and also thecombination of different signals can be individually adjusted by themixture of different functionalized carrier proteins and proteinswithout functional groups, so that the transfection agent according tothe invention can be designed as a modular system for the widest varietyof transfection conditions. The low extension in space and the highsignal density also make it possible to use in addition endogenoustransport systems which are specific for small molecules. Because of itsfilamentary character, its simple structure and its adjustable andextremely high signal density, the method according to the inventionmakes possible, or the transfection agent according to the invention iscapable of, specifically transporting of larger nucleic acid molecules,such as expression vectors, using endogenous mechanisms.

The additional functional components can for example be bound directlyto the NPF proteins or via a spacer, a non-functional separating unit.Spacers of this kind give rise to a greater distance between the NPF andthe functional components, which avoids mutual steric hindrance andwhich guarantees better spatial availability of the NPF protein and ofthe functional components. The structure of the agents according to theinvention thereby then largely avoids masking of the functionalcomponents.

NPF-forming proteins form nucleoprotein filaments with nucleic acidsmostly by cooperative binding, in which proteins and nucleic acids forma complex having a size or diameter that is much smaller than that ofthe known globular transfection agents. An NPF of this kind can forexample be formed by proteins of the RecA family, which areDNA-dependent ATPases, in the presence of nucleoside triphosphates suchas ATP (adenosine triphosphate), with total loading of a density of, forexample, one protein per three bases in double-stranded DNA (Bianco etal., 1998). This high protein density offers excellent protectionagainst enzymatic hydrolysis of the nucleic acids as the possible pointsof enzymatic attack are greatly reduced. The NPF not only causes thecomplex to be adequately stable but also allows adequate release of thetransported nucleic acids, for example, in the nucleus. Use ofnon-hydrolyzable or poorly hydrolyzable analogues of nucleosidetriphosphates, for example of ATP and/or GTP, such as ATP-γ-S orGTP-γ-S, also offers the possibility of providing extra stability toNPFs which are formed with ATP-forming proteins, such as the RecAfamily.

According to the invention, the proteins capable of forming NPFs alsoinclude derivatized NPF proteins. For example, fusion proteins can beproduced. In addition, NPF-forming proteins can be truncated orelongated, individual sections or amino acids can be deleted, introducedor chemically modified as long as their function which is essential tothe invention, the structural formation of NPFs with nucleic acids, ismaintained.

A particular advantage of the invention is the spatial structure of theNPFs which makes it possible to exploit the natural mechanisms ofnuclear transport. The maximal diameter of the transfection agent isdetermined by the size of the nuclear pores and is not exceeded evenwith very long nucleic acids, i.e. NPFs can be used as transfectingagents independently of the length of the nucleic acid to betransfected. It has been shown that the size limit for transport throughthe nuclear pore is approx. 25 nm (Feldherr and Akin, 1997) or 50 nm(SV40-Virus) (Yamada and Kasamatsu, 1993). Nucleic acids, of which theimport through the nuclear membrane with conventional transfectionagents having globular structure and/or non-specific binding of severalnucleic acid molecules per transfection component is barred, can easilylie under such limit using transfection agents according to theinvention. The structure of the transfection agents according to theinvention even makes it possible to use for the first time diameters of≦11 nm, depending on the NPF-forming protein used. The filamentousstructure assembled by the use of NPF proteins is therefore alsosuitable for the transport of longer nucleic acids of several kilobasesin length. The method according to the invention and the transfectionagents according to the invention are consequently particularly wellsuited for the transfection of cells with larger nucleic acid sequences.Transfection agents according to the invention are preferred whichcontain a nucleic acid to be transfected including at least 700nucleotides.

The present invention can advantageously be used as such for thetransfection of nucleic acids or in combination with other transfectionmethods and materials. The high degree of loading with NPF-formingproteins increases the stability to hydrolysis and the low diameter ofthe NPF allows the exploitation of endogenous cellular transportmechanisms which are only available to molecules which are small enough,for example, those of the nuclear transport system. In addition,adequate release of the nucleic acids to be transfected in the cellcompartments, preferably in the nucleus, is guaranteed.

The term “transfection agent” according to the invention is to beunderstood as transport vehicle for nucleic acids or their derivativeswhich already contain the nucleic acid to be transfected. A transfectionagent in the sense of the invention performs at least a single step ofthe complex process of transfection.

In the context of the present invention, the term “nucleoproteinfilament” (NPF) means a molecular structure consisting of nucleicacid(s) or nucleic acid derivatives and proteins which, as the result ofnon-covalent, mostly cooperative binding, form a filamentary orthread-like complex which preferably contains only a single nucleic acidmolecule or derivative of this. Particularly preferred are helicalnucleoprotein filaments, for example, those formed from RecA with singleor double-stranded DNA (Di Capua et al., 1982).

The “nucleic acid to be transfected” can be either a double or asingle-stranded DNA, or a double- or single-stranded RNA, or adouble-stranded DNA with single-stranded ends, a DNA/RNA hybrid, anantisense DNA, antisense RNA or chemically modified nucleic acidderivatives, in which, for example, the resistance to hydrolysis isincreased (peptide nucleic acid, PNA), or in which reactive moleculargroups have been introduced for the covalent binding and/or themodification of target nucleic acids. Derivatives of transfectablenucleic acids are understood to include the modification of the nucleicacid used with a sequence-specific or covalently bound protein. Thepreferred nucleic acid of the present invention is DNA, in particular,double-stranded DNA. Until now, NPF-forming proteins have mostly beenused together with single-stranded DNA, for example, for recombination.Surprisingly, it has turned out in the context of the invention thatmany NPF-forming proteins also form stable complexes withdouble-stranded DNA under suitable conditions and can be used for thetransfection of double-stranded DNA in accordance with the invention. Itis therefore a further advantage of the invention that efficienttransfection with double-stranded DNA is possible too.

In an advantageous embodiment of the invention, NPFs modified withfunctional components are produced by the modification of originalNPF-forming proteins or their derivatives. This modification can be thedeletion or insertion of amino acids and/or protein domains. Themodification can also be provided by chemical alteration of amino acidsand/or other molecular groups and/or by chemical coupling of peptides,proteins, carbohydrates, lipids or other molecules to the NPF-formingprotein or its derivative. These modifications may occur with the use ofa spacer.

The first hurdle for transfection consists in the association of thetransfection complex to the cell surface. A preferred embodiment of themethod and transfection agent according to the invention therefore usesat least one protein capable of forming NPF which is modified with atleast one functional group which causes the association of the complexor agent to the cell surface. This can be performed specifically for thecell type by binding to surface structures specific to the cell typewhich are expressed on one or only a few cell types, ornon-specifically, for example, by an electrostatic interaction. Allnaturally occurring or synthetically produced substances can be used forthe cell type-specific binding which bind to receptors on the cellsurface, such as receptors which are used by viruses or bacteria forcell entry, e.g. the Epstein Barr Virus receptor CD21 (Delcayre, 1991)or Listeria monocytogenes receptor E-cadherin (Mengaud, 1996). Otherexamples for the use of ligand-receptor pairs include the transferrinreceptor/transferrin system for cells which need a lot of iron,asialoglycoprotein receptor/galactose for hepatocytes,integrin/integrin-binding peptides, such as RGD (Harbottle 1998) ormolossin (Collins 2000) or hormones which bind to hormone receptors,such as insulin (Rosenkranz 1992), EGF (epidermal growth factor) orinsulin-like growth factor I (Feero, 1997) and oligosaccharides whichbind to lectins (Midoux et al., 1993). It is also possible to usecell-specific antibodies (Fominaya, 1996) or protein A or itsIgG-binding domain (Ohno, 1997) as well as biotinylated proteins (eitherproteins biotinylated on the cell surface or biotinylated monoclonalantibodies) in combination with streptavidin (Schoeman, 1995). It isparticularly suitable in the context of the present invention to use“epitope tagging”, i.e. cell type specific transfection usingbiospecific antibodies which, on the one hand can recognize an epitope,namely short peptides such as from the influenza hemagglutinin (Surdej,1994) or from the c-myc protein (Evan, 1985) which are either coupled orfused by genetic engineering to the NPF-forming proteins or theirderivatives and, on the other hand, to specific cell surface structures.For non-cell specific interactions of the transfection complex byelectrostatic forces it is possible, for example, to introduce positivecharges with additional amino acids (lysine, arginine, histidine).

In another particularly preferred embodiment of the method andtransfection agent according to the invention, it is intended that thefunctional components cause the non-endosomal passage of the complex oragent through the cell membrane. The non-endosomal membrane passage hasthe advantage that the agent is immediately available in the cytosol andis not exposed to the hydrolytically active environment of thelysosomes. Membrane passage of this kind can be attained withmembrane-active molecules. These can be naturally occurring, modified orsynthetic peptides which are mostly aliphatic or amphiphilic, such asviral peptides, for example HIV tat, VP22, HBV surface antigen; peptidesfrom transcription factors such as, for example, the homeodomain ofantennapedia (Thoren et al 2000), engrailed, HOXA-5; peptides fromcytokines, e.g. IL-1 β, FGF-1, FGF-2; peptides from cellular signalsequences, e.g. the Kaposi fibroblast growth factor, monoclonalantibodies which penetrate living cells, e.g. mab 3E10 (Weisbart et al2000), synthetic or chimeric peptides, e.g. amphiphilic model peptidesor transportane (Pooga et al. 1998).

In addition, an advantageous embodiment of the invention uses at leastone functional component according to the invention which causes therelease of the complex or agent from endosomes or lysosomes. Forexample, passage through the cell membrane can occur throughendocytosis. After endocytosis, the nucleoprotein complexes must bereleased from the endosomes. For this purpose, all substances withendosomolytic activity can be used. These can, for example, be peptides,their derivatives or synthetic analogues from bacteria or viruses, orother synthetic substances known to the person skilled in the art.Endosomolytic substances from bacteria include, for example,streptolysin O, pneumolysin, staphylococcal α-toxin, listeriolysin O(Provoda, 2000). Viral peptides include, for example, the N-terminalhemagglutinin HA-2 peptide of influenza virus (Steinhauer et al., 1995),the N-terminus of the VP-1 protein of rhinovirus HRV2 (Zauner et al.,1995) or the capsid component Ad2 of adenovirus (Hong, 1999). Syntheticsubstances include, for example, amphipathic peptides (GALA, KALA, EGLA,JTS1) (Wagner, 1999) or imidazole—(Pack, 2000) orpolyamidoamine-modified polymers (Richardson, 1999).

Passage (import) into the nucleus occurring usually in dependence oncell division and possibly after stimulation of the cell is a particularhurdle for all transfection agents. It is therefore intended in aparticularly advantageous embodiment of the invention to use afunctional component which causes the transport of the complex or agentinto the cell nucleus. The essentials in nuclear transport are firstlythe limiting diameter of the nuclear pores and secondly the signalmolecules used for transport. The signals for nuclear transport in thecontext of the invention are nuclear ligands which bind to a nuclearreceptor. Particularly suitable nuclear ligands are NLS (nuclearlocalization signals) or other components of the nuclear transportmachinery. The nuclear localization signals which are particularlypreferred for use are those signals which either themselves and/ortogether with their flanking regions exhibit little or no positivecharge excess, as a charge excess can lead to non-specific nucleic acidbinding and thus to masking of the signal. Well suited are extendedsequences of so-called classical NLS when the total charge of thepeptide can be at least approximately balanced by flanking negativelycharged amino acids. These amino acids can occur naturally in thepeptide/protein in these positions or be introduced there on the basisof structural considerations. The so-called non-classical NLS can alsobe used, for example, an NLS from the influenza virus “nucleoprotein”(Wang et al., 1997, Neumann et al., 1997) or the sequence M9 from theheterogenous nuclear RNP (hnRNP) A1 protein which have no great excessof positive charges or which pass into the nucleus by a non-classicaltransport route. An incomplete but good review of the NLS which can beused in the embodiment of the present invention is given by T. Boulikas(1993, 1996, 1997). Approximately charge-neutral sequences include, forexample, the large T-antigen from simian virus 40 having flankingnegative charges which are either naturally occurring or artificiallyintroduced (see also WO 00/40742 Amaxa).

A particular advantage of the invention is therefore the clearimprovement of the transfection of nucleic acids of eukaryotic cellswhich are either not dividing or only dividing weakly, in particularprimary eukaryotic cells. It is exactly these cells which have not yetforfeited their ability to provide biological or medical information, asthey can, for example, be taken directly from the body by blood samplingor tissue biopsy and are of decisive importance to the skilled person.With the expected analysis of the almost completely decoded humangenome, it is only the specific expression of the gene being examined incell systems which will finally give clear evidence of possibletechnical applicability with adequate physiological relevance. Inaddition, the transfection of primary cells is an essential preconditionfor non-viral gene therapy, both ex vivo and in vivo.

In an advantageous embodiment of the invention, the method andtransfection agent according to the invention can each include at leastone protein being capable of forming nucleoprotein filaments which ismodified with several functional components of different function and/ordifferent proteins which are modified with proteins of differentfunction, respectively. The transfection agents according to theinvention can, as described above, be realized with a plurality ofproteins which can form NPFs or derivatives thereof which may be presentin their original or in a functionally modified form. Either a single orseveral different proteins which can form NPFs may be used and these canbe modified with one or several functional components of the same ordifferent functions, depending on the specific requirements of thetransfection. Depending on the intended goal, different modules can beassembled by the user in this way. This gives rise to a modular systemfrom which unmodified or modified NPF proteins of the same or differentfunction can be selected in order to achieve an optimal adaptation ofthe transfection strategy to the nucleic acid to be transfected and thetarget cell.

A particularly advantageous embodiment of the invention is intended, inwhich the protein is loaded with a plurality of functional components.In this way, the total signal density can be still further increased, sothat improved transfection efficiency and better control of transfectionare possible.

Many NPF-forming proteins form the NPF structure in the presence ofnucleoside triphosphates, such as ATP, as cofactor. The addition ofnucleoside triphosphates hereby stabilizes the NPF. In the methodaccording to the invention, the NPF structure can therefore beadvantageously formed or stabilized by nucleoside triphosphates and/ornon-hydrolyzable analogues of these, in particular with ATP (adenosinetriphosphate) and/or GTP (guanosine triphosphate) and/or theirnon-hydrolyzable analogues. This can also occur by covalent bindingafter a photochemical reaction. Non-hydrolyzable nucleoside triphosphateanalogues include, for example, ATP-γ-S (adenosine5′-O-3-thiotriphosphate) and GTPγS (guanosine 5′-O-3-thiotriphosphate)(Ellouze, 1999). Possible ATP analogues which modify NPF-ATPases after aphotochemical reaction include 8N₃ATP (8-azidoadenosine 5′-triphosphate)and 5′FSBA (5′-p-fluorosulfonylbenzoyladenosine) (Knight, 1985).

In an advantageous embodiment of the method according to the invention,this is used in combination with other biological and/or chemical and/orphysical transfection methods for biologically active molecules, such asliposome-mediated transfer, microinjection, electroporation,immunoporation, the ballistic method, transfer with the help of cationiclipids, calcium phosphate, DEAE-dextran, polyethylenimine orpH-sensitive hydrogel. Transfer methods of this sort, in particularelectroporation, can advantageously support individual steps in thetransfection process, for example, such as the import into the cytoplasmof the cell.

Preferred transfection agents according to the invention include asNPF-forming protein a protein selected from the group of proteinscontaining RecA, RadA, ScRad51, RAD51, hDmc1, SASP, ICP8, preferablyUvsX, more preferably hRAD51 or a mixture of at least 2 of the proteinsin this group or one or several derivatives of these proteins.NPF-forming proteins for the embodiment of the present invention includefor example RecA from Escherichia coli and its functional homologuesfrom viruses, prokaryotes and eukaryotes, such as UvsX from thebacteriophage T4 (Mosig, 1987), RadA from archebacteria (Seitz et al.,1998), ScRad51 from Saccharomyces cerevisiae, RAD51 from mammals and, inparticular, hRad51 from man (e.g., variant 1, nucleotide and amino acidsequence described in NCBI accession no. NM_(—)002875; variant 2,nucleotide and amino acid sequence described in NCBI accession no.NM_(—)133487). Homologous proteins to RecA have been detected in atleast 60 different sorts of bacteria (Roca and Cox, 1990, Karlin andBrocchieri, 1996, Karlin et al., 1995), in archaea (Sandler et al.1996), in all eukaryotes which have been examined (Ogawa et al. 1993),in mitochondria (Thyagarajan et al., 1996) and in plastids (Cerutti etal., 1992). With single- or double-stranded DNA, RecA and its homologuesform helical NPF with a diameter of about 11 nm. The binding of RecA iscooperative and leads to a partial unwinding of the DNA helix. RecA,UvsX, ScRad51 and hRad51 form NPF with a binding stoichiometry of threebase pairs per monomer (double-stranded DNA) or 3-6 nucleotides permonomer (single-stranded DNA) (Bianco et al. 1998, Baumann and West1998). The meiosis-specific recombinase hDMC1 is preferred; this formsfilaments with double-stranded DNA which consist of a linear row ofstacked protein rings (Masson et al., 1999). In addition, the group ofSASP proteins (small acid-soluble spore proteins) from the spores ofBacillus and Clostridium species is also preferred. The SASPs also bindto double-stranded DNA forming helical NPF with a diameter of about 6.6nm (Griffith et al. 1994). Viral proteins which can form filaments withsingle- and/or double-stranded DNA, such as protein ICP8 from Herpessimplex, are also preferred as NPF-forming proteins (Lee & Knipe, 1985).

A binding stochiometry of one monomer protein per 3 base pairs has beendemonstrated for complete binding of the NPF-forming proteins UvsX,Rad51 and RecA to double-stranded DNA (Bianco et al 1998). Completebinding of this kindcan be attained with UvsX, for example, by using a3- to 5-fold excess of the protein, depending on the binding bufferused, the conformation of the nucleic acid and the temperature (Yu andEgelman 1993); with α/β type SASP from Bacillus subtilis thecorresponding protein:DNA ratio is about 5:1 (Griffith et al 1994). Itis particularly preferred in the context of this invention for theloading of the nucleic acid with protein to be as complete as at allpossible; this is defined separately for each of the preferredNPF-forming proteins. However, incomplete loading of the nucleic acidsbelow the absolutely highest degree of loading for a specificNPF-forming protein is possible in the context of the invention. Lowerloading of the nucleic acids with NPF-forming proteins is, for example,preferred when additional DNA-binding proteins are to be used fordefined steps in the transfection or when buffer conditions have to beselected for specific applications which are not optimal for completeloading of the nucleic acid with NPF-forming proteins.

According to the invention, NPF-forming proteins are understood toinclude both natural NPF-forming proteins and their derivatives. Underderivatives of NPF-forming proteins, the skilled person understandsfirstly modification by deletion or insertion of additional amino acidsequences or protein domains in recombinant proteins and/or theintroduction of functional groups by the chemical modification ofmolecular groups which are already present and/or the chemical couplingof proteins, peptides, carbohydrates, lipids or other molecules.

The transfection agent according to the invention can also beadvantageously used for producing drugs for the gene therapeutictreatment of humans and animals. The therapeutically useful nucleicacids can be made accessible to primary cells and complex transfectionmethods in the form of transfection agents according to the invention.

A further aspect of the present invention concerns pharmaceuticalpreparations which contain a transfection agent according to theinvention, possibly together with conventional adjuvants and carriers.

A further aspect of the present invention concerns kits which aresuitable for the transfection of cells with nucleic acids and whichinclude at least one protein capable of forming NPFs according to theinvention and at least one functional component according to theinvention as well as at least one of the following components:

a) nucleoside triphosphate and/or nucleoside triphosphate analogues

b) at least one nucleic acid to be transfected

c) adjuvants and additives

Such kits can be specifically designed for the different requirements ofexperts, for example, they can be optimized for certain nucleic acids,target cells or stability requirements, by the selection of specificNPFs, specific NPF modifications or individual adjuvants or additives.The protein can hereby already be loaded with the functionalcomponent(s) or the proteins or functional components can be containedseparately in the kit.

A further aspect of the present invention concerns the use of thetransfection agents according to the invention for cell screening,specifically for the identification of activators or inhibitors of theexpression product(s) of the transfection agent in mitotically inactiveor weakly active cells, primary cells and other cells of limited lifespan. Screening of this kind is a fundamental method for theidentification of activators or inhibitors of validated or non-validatedtarget proteins in the identification of active substances in thepharmaceutical industry. These screening methods are usually planned sothat cells which have been stably transfected with foreign nucleic acidsare exposed to potential inhibitors and/or activators and the influenceof these on the physiology of the cells is determined, possibly incomparison with comparator cells. The transfection agent according tothe invention is suitable for the insertion of the foreign nucleic acidshortly before the addition of activators or inhibitors, even inmitotically inactive or only weakly active cells, primary cells andother cells of limited life span, and thus to make these cellsaccessible as test cells.

A further aspect of the present invention concerns the use oftransfection agents according to the invention in the identification ofphysiologically active nucleic acids. This is of particular importancefor the rapid and physiologically relevant evaluation of genomic datawhich are available to the scientific and pharmaceutical community. Astransfection with the transfection agents according to the invention isindependent of cell division and/or endocytosis, the time betweentransfection and analysis is much shortened. This makes a much highersample throughput possible. Even cells which are difficult to transfectand even non-dividing cells are accessible with the transfection agentsaccording to the invention. The resulting transfection and thephysiological evaluation of changes in comparison to control cells makethe identification of physiologically active nucleic acids possible.

ABBREVIATIONS

Aside from the abbreviations usual in Duden¹, the followingabbreviations are used:

AMP-PCP Adenylyl-(β,γ-methylene)-diphosphonate

AMP-PNP S′-Adenylylimidodiphosphate

DEAE Diethylaminoethane

DNA Desoxyribonucleic acid

dYT Double yeast trypton

FACScan Fluorescence activated cell scanning

FCS Fetal calf serum

FL Fluorescence

FSC Forward scatter

GMP-PNP S′-Guanylylimidodiphosphate

GTP Guanosine triphosphate

H6 Histidine hexamer

Ig Immunoglobulin

kb Kilobases

ml Milliliter

mM Millimolar

msec Millisecond

NCBI National Center for Biology Information

ng Nanogram

nm Nanomolar

PBS Phosphate buffered salt solution

PCR Polymerase chain reaction

Pi Sodium dihydrogen phosphate/Disodium hydrogen phosphate

RNA Ribonucleic acid

RPMI Roswell Park Memorial Institute

SDS Sodium dodecylsulfate

SV40 Simian virus 40

TAE Tris-acetate/ethylendiaminetetraacetate

U/mg Units/Milligram

rpm Revolutions per minute

Cl-Puffer Cell injection buffer

μg Microgram

μl Microliter ¹ Translator's Note: German Standard Dictionary

SHORT DESCRIPTION OF THE FIGURES

FIG. 1: Schematic Representation of the Expression Plasmids

FIG. 1 shows a schematic representation of the structure and productionof the NPF-forming proteins described in the examples.

FIG. 2: Binding of UvsX to Double-Stranded DNA

FIG. 2 shows reaction mixtures of 200 ng (in each case) of a purified 1kb PCR fragment with the given quantities of H6UvsX in 76 mM K₂HPO₄, 17mM KH₂PO₄, 14 mM NaH₂PO₄, pH=7.2, 5 mM MgCl₂ and 0 or 1 mM ATP-γ-S,which was incubated in a final volume of 15 μl for 30 min at roomtemperature and then applied to a 0.8% TAE/agarose gel, which wasafterwards stained with ethidium bromide.

FIG. 3: Binding of NLS-modified UvsX to double-stranded DNA in thepresence of different ATP analogues

250 ng of a purified 1 kb PCR fragment were incubated with 15 μgUvsXH6N2 in 76 mM K₂HPO₄, 17 mM KH₂PO₄, 14 mM NaH₂PO₄, pH=7.2, 5 mMMgCl₂ and 0.5 or 2 mM of the given nucleotide analogue in a final volumeof 20 μl for 30 min at room temperature. The reaction mixture was thensplit and 220 ng of a 1.7 kb PCR fragment was added to one half (B),incubated for a further 30 min at room temperature; all reactionmixtures were then applied to a 0.8% TAE/agarose gel, which wasafterwards stained with ethidium bromide.

FIG. 4: Binding of a mixture of UvsX and modified UvsX todouble-stranded DNA

200 ng (in each case) of a purified 1 kb PCR fragment was incubated in76 mM K₂HPO₄, 17 mM KH₂PO₄, 14 mM NaH₂PO₄, pH=7.2, 5 mM MgCl₂ and 1 mMATP-γ-S with the given quantities of purified H6UvsX or UvsXH6N2 for 30min at RT and then all reaction mixtures were applied to a 0.8%TAE/agarose gel, which was afterwards stained with ethidium bromide. Toexclude the possibility that the electrophoretic behavior of DNA isartificial as a result of salt, traces of imidazole or glycerine,samples of elution or dialysis buffer were included in tracks 8 and 9.(Final concentration: 1/10 vol elution buffer with 500 mM imidazole,3/20 volumes dialysis buffer with 50% glycerine).

FIG. 5: FACScan Analysis of the Transfection of NIH3T3 Cells inCombination with Electroporation

FIG. 5 a-d shows an FACScan analysis: 5 a) electroporation without DNA,5 b) electroporation of vector DNA without UvsX, 5 c) with vector DNApackaged in UvsX, 5 d) with vector DNA packaged in UvsX-NLS

FIG. 6: A Further FACScan Analysis of the Transfection of NIH3T3 Cellsin Combination with Electroporation

FIG. 6 shows a bar diagram of the results of an FACSscan analysis oftransfection with vector DNA packaged in UvsX-NLS (UvsXH6N2-2) andvector DNA packaged in UvsX “scrambled” NLS (UvsXH6N2sc).

FIG. 7: Fluorescence Microscopic Analysis of the Transfection of NIH3T3Cells in Combination with Microinjection.

The expression of a fluorescent marker protein (left side) is shown inmicroinjected NIH3T3 cells. BSA-Cy5 serves as injection marker, which isvisualized in the corresponding fluorescence filter (right side).Pictures 1 and 2 show cells which were injected with DNA and UvsXH6N2scin the cytoplasm; Pictures 3 and 4 show cells which were injected withDNA and UvsXH6N2-2; Pictures 5 and 6 show cells that were only injectedwith DNA.

FIG. 8: Schematic Representation of the Expression Plasmids for SASPProteins

FIG. 8 shows a schematic representation of the structure and productionof the SASP proteins described in Examples 7 and 8.

FIG. 9

-   -   (A) Purification of SASP Protein    -   (B) DNA Binding Ability of SASP Protein

FIG. 9A shows an SDS gel with purified SASP protein. FIG. 9B shows thebinding of DNA by SASP protein in a DNA shift analysis.

FIG. 10

-   -   (A) Purification of SASP-NLS-Protein    -   (B) DNA Binding Ability of SASP-NLS-Protein

FIG. 10A shows an SDS gel with purified SASP-NLS-Protein (SASP-H6N2).

FIG. 10B shows the binding of DNA by SASP-NLS-Protein in a DNA shiftanalysis.

FIG. 11: Coupling of a Peptide with RGD-Motive to H6UvsX

FIG. 11 shows the electrophoretic separation of H6UvsX protein with andwithout the chemically coupled peptide RGD2. Two independently preparedsamples of H6UvsX*(RGD2) are shown. 1 μg protein was applied in eachtrack.

FIG. 12: Binding of Differently Modified UvsX Proteins toDouble-Stranded DNA

FIG. 12 shows the separation in agarose gels of the NPFs consisting of adouble-stranded DNA fragment and a mixture of H6UvsX*(RGD2) andUvsXH6N2-2 (Track 1), or of UvsXH6N2-2 alone (Track 2) or ofH6UvsX*(RGD2) alone (Track 3).

FIG. 13: DNA Shift Analysis of the Binding of UvsXH6N2 and UvsXH6N2NIT-2to Fluorescein-Labeled DNA

FIG. 13 shows a DNA shift of DNA-UvsXH6N2 and DNA-UvsXH6N2NIT-2. Theproteins were incubated with a AlexaFluor488-labeled DNA fragment, asdescribed in Example 10.

FIG. 14: Fluorescence Microscopy Analysis of the Uptake of NPFs inNIH3T3 Cells by Endocytosis

FIGS. 14 a and 14 b show fluorescence microscopy pictures of NIH3T3cells. In each case, one picture shows a bright field image (bottom) andone in reflected light fluorescence (top). In the bright field,vesicular intracellular compartments are recognizable, which emitfluorescent light because of the endocytosed fluorescein-labeled DNA.

FIG. 15: Percentage of Cells with Endocytosed NPFs

FIG. 15 is a bar diagram which shows the percentage of the cells whichinhibit at least one fluorescent vesicular compartment.

FIG. 16: Schematic Representation of the Expression Plasmids

FIG. 16 shows the schematic representation of the structure of theexpression plasmids for the production of hRad51 fusion proteins.

FIG. 17: Binding of NLS-Modified hRad51 (hRad51H6N2) to Double-StrandedDNA

FIG. 17(A) shows an SDS/Coomassie gel with hRad51H6N2 (12.7 μg) andhRad51H6 (11 μg) after purification over nickel-chelate affinitychromatography. FIG. 17(B) shows reaction mixtures of 100 ng (in eachcase) of a purified 0.9 kb PCR fragment with the given quantities ofhRad51H6N2 in 38 mM K₂HPO₄, 8.5 mM KH₂PO₄, 7 mM NaH₂PO₄, pH=7.2, 15 mMMgCl₂, 2.5 mM ATP and 25% glycerine, which were incubated in a finalvolume of 30 μl for 10 min at 37° C. and then applied to a 1%TAE/agarose gel which was afterwards stained with ethidium bromide.

FIG. 18 Binding of a Mixture of Differently Modified UvsX (UvsX-NLS-VP22and UvsX-NLS) to Double-Stranded DNA

FIG. 18(A) shows a SDS/Coomassie gel with UvsXH6N2VP22c50.

FIG. 18(B): 140 ng (in each case) of a purified 1.7 kb PCR fragment wereincubated in 96 mM K₂HPO₄, 21.5 mM KH₂PO₄, 18 mM NaH₂PO₄, pH=7.2, 5 mMMgCl₂ and 1.3 mM ATP-γ-S with the indicated quantities of purifiedUvsXH6N2VP22c50 or UvsXH6N2-2 for 30 min at RT. All reaction mixtureswere then applied to a 0.8% TAE/agarose gel, which was afterwardsstained with ethidium bromide.

FIG. 19: Transfected NIH3T3 Cells after Treatment with Complexes of DNAand a Mixture of UvsX-NLS-VP22 and UvsX-NLS

Fluorescence microscopy picture of NIH3T3 cells, 24 h after treatmentwith complexes of 1 μg expression vector DNA and a mixture of 19 μgUvsXH6N2VP22c50 and 39 μg UvsXH6N2-2.

FIG. 20: Schematic Representation of the Basic Structure of the Methodand Transfection Agent based on Nucleoprotein Filaments, according tothe Invention.

DESCRIPTION OF THE INVENTION

The following examples are intended to describe the invention in detail,without limiting it to exemplary disclosed substances and methods.

Example 1 Generation of Recombinant UvsX as NPF Forming Proteins

The proteins UvsXH6, UvsXH6-2, H6UvsX und H6UvsX-2 were used as NPFforming proteins (see FIG. 1).

Structure of the Proteins:

UvsXH6 (400 amino acids):

Amino acids 1-391: UvsX from the phage T4 (NCBI protein accession no:AAD42669, amino acids 1-391), amino acids 392-394: linker consisting ofthe amino acids G³⁹²GS³⁹⁴, amino acids 395-400: H³⁹⁵HHHHH⁴⁰⁰ forpurification by nickel chelate affinity chromatography, amino acidexchange: L⁴³→P.

UvsXH6-2 (403 amino acids):

Amino acids 1-391: UvsX from the phage T4 (NCBI protein accession no:AAD42669, amino acids 1-391), amino acids 392-394: linker consisting ofthe S³⁹²YG³⁹⁴, amino acids 395-400: H³⁹⁵HHHHH⁴⁰⁰, amino acids 401-403:C-terminus consisting of the amino acids M⁴⁰¹YS⁴⁰³

H6UvsX (404 Amino Acids):

Amino acids 1-4: N-terminus consisting of the amino acids M¹SYS⁴, aminoacids 5-10: H⁵HHHHH¹⁰, amino acids 11-13: linker consisting of the aminoacids S¹¹YG¹³, amino acids 14-404: UvsX from the phage T4 (NCBI proteinaccession no: AAD42669, amino acids 1-391), amino acid exchange: Q³⁴⁰→L.

H6UvsX-2 (404 Amino Acids):

Amino acids 1-4: N-terminus consisting of the amino acids M¹GYS⁴, aminoacids 5-10: H⁵HHHHH¹⁰, amino acids 11-13: linker consisting of the aminoacids S¹¹YG¹³, amino acids 14-404: UvsX from the phage T4 (NCBI proteinaccession no: AAD42669, amino acids 1-391).

Cloning of the Expression Plasmids

For the expression of the aforementioned proteins in suitableEscherichia coli cells, plasmids were constructed containing a codingsequence for UvsXH6, UvsXH6-2, H6UvsX or H6UvsX-2 under the control ofthe lac promotor (pExH-UvsXH6, pExH-UvsXH6-2, pExH-H6UvsX orpExH-H6UvsX-2, see FIG. 1). The plasmids were generated by ligation oftwo PCR products. The first PCR product was amplified from pMCS5(MoBiTec, Göttingen, Germany). pMCS5 is constructed in a similar way aspBluescript SK(−) (Stratagene) and is only different in the 5′ region ofthe coding sequence of the lacZα fragment. PMCS5 therefore contains thelac promotor followed by the lac operator, by which the expression of aninserted coding sequence is based on the absence of active lacrepressor. In order to achieve constitutive expression in any case, theamplification primers were selected in such way, that the lac operatoris not present anymore in the PCR product. The resulting PCR productcorresponded to pMCS5 from position 992 to 664 plus restrictionoverhangs. The nucleotides GAATTC (EcoRI restriction site) as well asTGTGTG were added before position 992 (3′ to the lac promotor), and thenucleotides ACTAGT (Spe I restriction site) and CACACA were added behindposition 664 to enable the ligation after digestion with therestrictions enzymes EcoRI and SpeI. The coding sequence for UvsXH6,UvsXH6-2 or H6UvsX and H6UvsX-2 was obtained by PCR amplification of T4DNA using primers which contain the desired restriction sites, aribosomal binding site and the additional codons. At their 5′ end beforethe start codon, the PCR products contained the additional nucleotides5′-CACACAGAATTCATAAAGGAAGATATCAT-3′ (SEQ ID NO:2), as well as theadditional nucleotides 5′-ACTAGTTGTGTG-3′ (SEQ ID NO:3) at their 3′ endafter the stop codon.

Purification:

An overnight culture of pExH-H6UvsX in DH5 was inoculated with 1.5-3 ldYT/ampicillin (200 μg/ml) 1:1000, and was grown over night at 37° C.with 250 UPM. The cultures were harvested at 7000×g, and yielded approx.5-15 g bacteria sediment. This was frozen for 1-3 days at −20° C. Thesediment was thawed on ice and resuspended in 10-20 ml cold startingbuffer. The lysis was carried out under slow stirring for 1 h at 4° C.using 10 ml lysozym (Serva, 190.000 u/mg) and approx. 4 g glass beads(Sigma, G-8893). Then, 50 μl DNAse I (Serva, 2 mg/ml) were added andfurther incubated for another 30 min. After centrifugation of the lysate(45 min, 11.000×g, 4° C.), the supernatant was filtered through asterile filter (pore size 0.45 μm) and loaded on an equilibrated 1 mlHiTrap™ Chelating column (Pharmacia) preloaded with Ni⁺⁺ ions. Thefurther purification steps were carried out according to the respectivePharmacia protocol for proteins containing a histidin hexamer. Aliquotsof the various elution fractions were added to SDS/Coomassie gels. Thepurest fractions were combined and further concentrated in CentriplusYM30 columns (Millipore) according to the respective protocol. Then, itwas dialyzed twice (dialysis tube: Spectra/Por, MWCO: 25.000) against anat least one thousand-fold volume of ZI buffer for at least one hour at4° C., then over night at 4° C. against ZI buffer/50% glycerine. Thedialysate was aliquoted in 30-50 μl fractions and stored at −80° C.

Used Buffers:

ZI buffer: 76 mM K₂HPO₄, 17 mM KH₂PO₄, 14 mM NaH₂PO₄, pH=7.2

Starting buffer: 20 mM Pi, 0.5 M NaCl, 10 mM imidazole, pH=7.4

Washing buffer: 20 mM Pi, 0.5 M NaCl, 20-50 mM imidazole, pH=7.4

Elution buffer: 20 mM Pi, 0.5 M NaCl, 100-500 mM imidazole, pH=7.4

Determination of Concentration:

The concentration of the UvsX proteins was determined by measuring theOD₂₈₀ using the extinction coefficient calculated with the GeneInspector™ (Textco, Inc.) software. For H6UvsX it was 2.5-3.5 μg/11.

Description of the Experiment:

H6UvsX, purified over Ni⁺⁺ sepharose, was incubated with 200 ng of a 1kb PCR fragment (with and without 1 mM ATP-γ-S).

A shift of the DNA in the agarose gel caused by protein binding shows,that H6UvsX binds double-stranded DNA based on the concentration, andthat this binding is enhanced by ATP-γ-S.

With ATP-γ-S, protein-DNA complexes are formed which can be stained moreintensely with ethidium bromide than without ATP-γ-S, which indicates atopological change of the nucleoprotein filament by the nucleotideanalog.

Example 2 Generation of a Transfecting Agent, Based on UvsX as NPFForming Protein with a Nuclear Localization Signal as a FunctionalComponent

As in example 1, plasmids were generated which permit the expression ofthe fusion proteins UvsXH6N2, UvsXH6N2-2, N2H6UvsX and N2H6UvsX-2, whichare based on the proteins described in example 1, and in additioncontain a nuclear localization signal (see FIG. 1).

Structure of the Proteins:

UvsXH6N2 (426 amino acids):

Amino acids 1-391: UvsX from the phage T4 (NCBI protein accession no.:AAD42669, amino acids 1-391), amino acids 392-394: linker consisting ofthe amino acids G³⁹²GS³⁹⁴, amino acids 395-400: H³⁹⁵HHHHH⁴⁰⁰, aminoacids 401-403: linker consisting of the amino acids G⁴⁰¹ GS⁴⁰³, aminoacids 404-417: nuclear localization signal nls-2 (amino acids 2-15, SEQID NO: 9 from WO 00/40742), amino acids 418-421: C-terminus of UvsX fromthe phage T4 (NCBI protein accession no: AAD42669, amino acids 388-391),amino acids 422-426: C-terminus consisting of the amino acids K⁴²²LVTG⁴²⁶, amino acid exchange: Y²³⁸→V.

UvsXH6N2-2 (420 amino acids):

Amino acids 1-391: UvsX from the phage T4 (NCBI protein accession no:AAD42669, amino acids 1-391), amino acids 392-394: linker consisting ofthe amino acids S³⁹² YG³⁹⁴, amino acids 395-400: H³⁹⁵HHHHH⁴⁰⁰, aminoacids 401-403: linker consisting of the amino acids M⁴⁰¹S⁴⁰³, aminoacids 404-417: nuclear localization signal nls-2 (amino acids 2-15, SEQID NO: 9 from WO 00/40742), amino acids 418-420: C-terminus consistingof the amino acids G⁴¹⁸YP⁴²⁰.

N2H6UvsX (420 amino acids):

Amino acids 1-3: N-terminus consisting of the amino acids M¹SY³, aminoacids 4-17: nuclear localization signal nls-2 (amino acids 2-15, SEQ IDNO: 9 from WO 00/40742), amino acids 18-20: linker consisting of theamino acids L¹⁸YS²⁰, amino acids 21-26: H²¹HHHHH²⁶, amino acids 27-29:linker consisting of the amino acids S²⁷YG²⁹, amino acids 30-420: UvsXfrom the phage T4 (NCBI protein accession no: AAD42669, amino acids1-391), amino acid exchange: Q³⁵⁶→L.

N2H6UvsX-2 (421 amino acids):

Amino acids 1-4: N-terminus consisting of the amino acids M¹GYP⁴, aminoacids 5-18: nuclear localization signal nls-2 (amino acids 2-15, SEQ IDNO: 9 from WO 00/40742), amino acids 19-21: linker consisting of theamino acids S¹⁹YS²¹, amino acids 22-27: H²²HHHHH²⁷, amino acids 28-30:linker consisting of the amino acids S²⁸YG³⁰, amino acids 31-421: UvsXfrom the phage T4 (NCBI protein accession no: AAD42669, amino acids1-391).

Cloning of the Expression Plasmids:

For the expression in suitable Escherichia coli cells, plasmids wereconstructed containing a coding sequence for UvsXH6N2, UvsXH6N2-2,N2H6UvsX or N2H6UvsX-2 under the control of the lac promotor(pExH-UvsXH6N2, pExH-UvsXH6N2-2, pExH-N2H6UvsX or pExH-N2H6UvsX-2, seeFIG. 1). The plasmids were generated as described in example 1 byligation of two PCR products (see FIG. 1).

Purification:

The purification of UvsXH6N2 was carried out as described in example 1)for H6UvsX.

Concentration: 10-20 μg/11.

Description of the Experiment:

UvsXH6N2 binds to double-stranded DNA. The binding is stabilized byATP-γ-S:

In order to examine the influence of various ATP analogues on thebinding performance of UvsXH6N2 to DNA, the protein was first incubatedwith a 1 kb DNA fragment and various ATP analogues. Then, a 1.7 kb DNAfragment was added for competition. If the binding of the protein to theDNA is stabilized by addition of an ATP analog, it can be expected thatUvsXH6N2 less likely binds a competing DNA fragment as long as noequilibrium is present. As can be seen in FIG. 3, the protein-DNAcomplex which was generated with the 1 kb fragment and UvsXH6N2, remainsstable in absence of ATP-γ-S and the 1.7 kb fragment, compared to allother used ATP analogues, i.e. the 1.7 kb fragment apparently is not oronly marginally occupied by liberated UvsXH6N2 molecules within theobservation time.

Example 3 Generation of a Transfection Agent with a Mixture of Modifiedand Unmodified NPF-Forming Protein

Description of the Experiment:

Various ratios of H6UvsX and UvsXH6N2 were incubated with a 1 kb DNAfragment. The two proteins retarded the DNA in different degrees, due totheir different molecular weight (see column 2 and 3 of FIG. 4). If theproteins are mixed before addition of DNA, intermediate complexes areformed based on the ratio of H6UvsX to UvsXH6N2, which yield a sharpband and therefore have an equal mean molecular weight. This shows thatthe DNA is statistically equally occupied by both proteins.

Example 4 Transfection of a Cell Line (NIH3T3) with UvsX-NLS inCombination with Electroporation

NIH3T3-Zellen (adherent, cultivated until 70-80% confluent) weretransfected with a vector coding for the heavy chain of the murine MHCclass I proteins H-2K^(K). 1×10⁶ cells were electroporated with 25 ngvector DNA which has been preincubated in binding buffer (76 mM K₂HPO₄,17 mM KH₂PO₄, 14 mM NaH₂PO₄, 5 mM MgCl₂ pH 7.21) with 14 μg UvsX orUvsX-NLS as well as with or without 1 mM ATP-γ-S for 30 minutes at roomtemperature. For this, the cells were added to a total volume of 100 μlelectroporation buffer (103 mM NaCl, 5.36 mM KCl, 0.41 mM MgCl₂, 23.8 mMNaHCO₃, 5.64 mM Na₂HPO₄, 11.2 mM glucose, 0.42 mM Ca(NO₃)₂, 20 mM HEPES,3.25 μM gluthathione) and electroporated in a cuvette with 2 mmelectrode spacing. The electroporation was carried out by an exponentialdischarge at a voltage of 240 V and a capacity of 450 μF. The half-lifeof the voltage drop was typically 12 msec. Immediately after theelectroporation, the cells were flushed out of the cuvettes with culturemedium (RPMI with 10% FCS), incubated for 10 min at 37° C., and thentransferred to a culture dish with prewarmed culture medium. After 6 hincubation, the cells were harvested and washed twice with PBS, and thenincubated with a Cy5-coupled anti-H-2K^(K) antibody and analyzedflow-cytometrically (FACScan). The number of dead cells was determinedby staining with propidium iodine. Six hours after the electroporation,7.4% or 8.7% of the cells transfected with free vector DNA express theH-2K^(K) protein (minus the background of 0.25% in average). Incomparison, the expression rate of the cells that have been transfectedwith vector UvsX was 2.9% or 3.8%. The expression rate after atransfection with vector UvsX-NLS was 19.2% or 18.9% (see FIGS. 5 a-5d).

For examination of the nuclear transport, the physical procedure ofelectroporation was chosen so that no other biochemical componentsexcept UvsX influence the transfection. The tight binding of UvsX withATP-γ-S to DNA, however, impairs the mobility of the complex in theelectric field and therefore reduces the efficiency of theelectroporation by approx. 60%. The attachment of a nuclear transportsignal alone results in an increase of the expression shortly after thetransfection by a factor of averagely 5.7 in this system. The increaseof the expression rate by UvsX-NLS compared to UvsX thereforedemonstrated that using a nuclear transport signal as functionalcomponent, DNA is transported into the nucleus by UvsX. An analysisperformed shortly after the transfection is significantly improved sinceeven cells become accessible that have not divided between transfectionand analysis.

Example 5 Transfection of a Cell Line (NIH3T3) with UvsX-Scrambled-NLSor UvsX-NLS in Combination with Electroporation

Generation of UvsX-Scrambled-NLS

In order to test the influence of the nuclear localization signal on thetransfection, an UvsX derivative was generated for comparison purposeswhich corresponds in its net charge to an UvsX protein with a NLS, butitself does not contain a functional NLS.

Using partly homologous oligonucleotide primers, the UvsX gene wasamplified from plasmid pExH-UvsXH6N2-2 (compare FIG. 1), so that theamino acid sequence SEQ ID NO:1 (“scrambled”, i.e. a mixed NLS sequence)is expressed at the C-terminus of the resulting protein UvsXH6N2scinstead of the amino acid sequence EEDTPPKKKRKVED (SEQ ID NO:4)(“nls-2”, corresponding to the amino acids 2-15 from SEQ ID NO:9 from WO00/40742). The scrambled amino acids correspond to the described nls-2with respect to their composition but not with respect to their order.The net charges of UvsXH6N2-2 and UvsXH6N2sc are therefore equal, butonly UvsXH6N2-2 contains an intact nuclear localization signal.

The protein UvsXH6N2sc was purified as described for the proteins inexample 1.

NIH3T3 cells (adherent, cultivated until 70-80% confluent) weretransfected with this vector coding for a fluorescent marker protein.For this purpose, 25 ng vector DNA in binding buffer (see example 1)were initially incubated with 1.5 mM ATP-γ-S and 16-18 μg of thedescribed proteins for 30 min at room temperature. The protein-DNAcomplexes were each added to 3×10⁵ NIH3T3-Zellen, resuspended in 80 μlelectroporation buffer (140 mM Na₂HP₄/NaH₂PO₄, 10 mM MgCl₂, 5 mM KCl, pH7.2). The electroporation was carried out in a cuvette with 2 mmelectrode spacing by an exponential discharge at a voltage of 240 V anda capacity of 450 μF, The half life of the voltage drop was typically 12msec. After addition of 400 μl medium, composed of RPMI 1640, Gibcocompany, 5% FCS, 2 mM glutamax (L-alanyl-L-glutamine, Invitrogen), 100U/ml penicillin/streptomycin, 0.5 mM β-mercaptoethanol, the cells wereadded to culture dishes (6-well plates) with 1 ml prewarmed medium andwere incubated at 37° C. and 5% CO₂. After 6 h, the flow-cytometricanalysis was carried out (FACScan).

The result is graphically shown in FIG. 6: 9% of the cells transfectedwith free vector DNA express the marker protein. The expression rate ofthe cells that have been transfected with vector-UvsX-NLS(DNA+UvsXH6N2-2), however, was 21%. In comparison, the expression rateof the cells after transfection with vector-UvsX-scrambled-NLS(DNA+UvsXH6N2sc) was only 4%.

UvsX, modified with a nuclear localization signal therefore results in amarkedly increased efficiency compared to free DNA and also compared tothe modification by a non-functional nuclear localization signal. Sincethe latter transfection represents the control, this means that thetransfection efficiency could be increased to 5-fold by modification ofthe UvsX. Therefore, the transfection efficiency can be markedlyincreased by the method according to the invention or the transfectionagent. Furthermore a targeted control of the transfection method, here,for example, a directing into the nucleus, is possible in anadvantageous way when the NPF-forming protein is modified (see alsoexample 6).

Example 6 Transfection of a Cell Line (NIH3T3) in Combination withMicroinjektion

140 ng of a 1.7 kb expression vector DNA fragment were incubated with 9μg modified UvsX protein as described above in binding buffer and 1 mMATP-γ-S in a final volume of 20 μl for 30 min at RT. BSA-Cy5 was used asinjection marker immediately before the injection in a concentration ofapprox. 1 μg/μl.

NIH3T3 cells that had been seeded to subconfluency the day before onCELLocate coverslips (Eppendorf) were microinjected through samplesloaded onto Femtotipps (Eppendorf) using a micromanipulator andtransjector (Eppendorf) under an inverse fluorescence microscope (LeicaDMIL).

The analysis was carried out in a fluorescence microscope (Olympus BX 60fluorescence microscope, digital b/w camera SPOT-RT from DiagnosticInstruments Inc., analysis software: Metaview Imaging System fromUniversal Imaging Corporation) after 5 hours of further incubation ofthe cells at 37° C. and 5% CO2. FIG. 7 shows microinjected NIH3T3 cells.The images 1 and 2 show cells that were injected with DNA and UvsXH6N2scinto the cytoplasm, the images 3 and 4 show cells that were injectedwith DNA and UvsXH6N2-2, and images 5 and 6 show cells that wereinjected only with DNA. Expression was only observed when theprotein-DNA complexes contained UvsXH6N2-2, i.e. an UvsX proteinmodified with a nuclear localization signal (FIG. 7, image 3). In thecells of the controls (FIG. 7, image 5: only DNA, or FIG. 7, image 1:DNA with UvsX-scrambled-NLS) that had been clearly injected only in thecytoplasm and not in the nucleus during microinjection, no expressionwas observed, not even using a very long exposure.

Example 7 Generation of Recombinant SASP as NPF-Forming Proteins

Cloning, Expression and Purification of the SASP Protein from B.subtilis

The SspC gene from Bacillus subtilis, which codes for a SASP (“smallacid-soluble spore protein”), was synthesized from 8 oligonucleotidesaccording to the Khorana method (described in Bertram and Gassen:Gentechnische Methoden, Gustav Fischer Verlag, 1991, p. 212-213) andligated between the NcoI and BglII restriction sites of the plasmidpARA13 (Cagnon et al., 1991; Protein Engng. 4: 843-847). This was basedon the protein sequence with the NCBI protein accession no:NP_(—)389876. The reverse transcription to DNA was carried out using thecodon preferences of genes, strongly expressed in E. coli and describedin (Andersson and Kurland 1990; Microbiol. Rev. 54: 98-210). Theresulting plasmid pARA13-SASP served as a matrix for the cloning of twoother plasmids, which code for SASP proteins, carrying a polyhistidinesequence of 6 histidines either at the N-terminus (H6-SASP) or at theC-terminus (SASP-H6) (FIG. 8). The plasmids were transformed into the E.coli strain BL21 (DE3) pLysS (Novagen, Madison) and plated out onLB/ampicillin/glucose (0.2%).

20 ml M9 minimal medium (0.2% glucose) each was inoculated with singlecolonies and grown over night at 37° C. and 220 rpm. The following day,the cultures were induced with 0.2% arabinose at an OD₆₀₀ of approx. 1.0and were grown for another 6 hours. Raw extracts (0.5 ml pelletedculture in PBS/loading buffer) were applied to high resolution SDS gels(according to Schägger and von Jagow 1987; Anal. Biochem. 166:368-79)and stained with Coomassie Blue (FIG. 9A).

For preparative purification, 2 L M9/glucose were inoculated 1:200 withan overnight culture. Again, the culture was induced with 0.2% arabinoseat an OD₆₀₀ of approx. 1.0, and further grown for 6 h. The bacteria werethen pelleted and frozen at −20° C. In the following, the purificationof SASP-H6 is described exemplary. Approx. 7 g of the pellet was thawedand resuspended in 14 ml starting buffer (see example 1), supplementedwith a tablet of complete EDTA-free protease inhibitor cocktail, Roche,Mannheim, and sonificated on ice for 3 min at 280 Watts (Labsonic U,Braun Biotech, Melsungen repeating duty puls; 0.5 sec). The extract wascentrifuged at 4° C. Apart from this, the purification was carried outas described for the UvsX proteins over HiTrap chelating columns(Amersham Pharmacia, Uppsala). The fractions between 200 mM and 500 mMimidazole, containing the protein in high concentration, were combinedand concentrated to approx. 3 ml using centriplus columns (YM-3,Millipore, Eschborn). The SASP protein was then dialyzed three timesagainst 1×ZI buffer (see example 1), and aliquots were frozen at −80° C.in a concentration of approx. 5 μg/11.

Testing of the DNA-Binding Capacity of the SASP Protein

125 ng (in each case) of a 1.7 kb DNA fragment were preincubated withdifferent amounts of SASP-H6 protein in 1×ZI buffer or 1/10×ZI bufferfor 30 min at room temperature, and then applied to an 0.8% TAE-agarosegel.

FIG. 9B shows that the DNA is completely bound by the protein. Thediffuse appearance of the DNA-protein bands may be due to a dissociationof the proteins from the DNA during the gel run.

Example 8 Generation of a Transfection Agent Based on SASP asNPF-Forming Protein with a Nuclear Localization Signal as Modification

For generation of an SASP with a nuclear localization signal (NLS) asfunctional component, a DNA sequence coding for a nuclear localizationsignal (“nls-2”, amino acids 2-15, SEQ ID NO: 9 from WO 00/40742) wasadded to the C-terminus of the already available clone pARA13-SASP-H6(see FIG. 8) by PCR amplification. The aforementioned plasmid served asPCR template. The resulting plasmid pARA13-SASP-H6N2 (see FIG. 8) wastransformed as described in example 7, and the protein SASP-H6N2 waspurified accordingly.

Testing of the Binding Ability of the SASP-NLS Protein

125 ng each of a 1.7 kb DNA fragment were preincubated with differentamounts of SASP-H6N2 protein in 1×ZI-Puffer for 30 min at roomtemperature, and then applied to a 0.8% TAE-agarose gel.

FIG. 10B shows that the DNA is held back by the NLS-modified SASP aswell. The DNA protein bands appear diffuse.

Example 9 Generation of a Transfection Agent with a Integrin BindingMotif for the Association of the Complex to the Cell Surface asFunctional Component

Integrins are membrane-anchored adhesion proteins on the cell surfacesome of which recognize a peptide motif of three amino acids(arginine-glycine-aspartic acid or “RGD” motif) as binding partner. Thebinding results in clustering of several integrin molecules on the cellsurface and in endocytosis (Plow et al., 2001). By modification of UvsXas NPF-binding protein with an RGD motif, it can be achieved that thetransfection agens can be taken up specifically via integrins into theendosomal compartments of the cells.

Structure of the Proteins

The used proteins H6UvsX and UvsXH6N2-2 are identical to the ones shownin FIG. 1 and described in example 1 and 2.

The protein H6UvsX*(RGD2) was generated by chemical coupling. Theprotein named RGD2 is a nonapeptide (NITRGDTYI) consisting of the pentonbase protein of adenovirus type 7 (Bal et al., 2000), which has beensynthesized in such way that a chemically active group is present at theN-terminal amino group (SMCC, succinimidyl4-[N-maleimidomethyl]-cyclohexane-1-carboxylat) which permits thecoupling to free cysteine SH groups in the UvsX protein.

For the coupling, 6 nmol H6UvsX were incubated with 60 nmol peptide in76 mM K₂HPO₄, 17 mM KH₂PO₄, 14 mM NaH₂PO₄, pH=7.2 for one hour at 37°C., and was then purified by several washing steps with incubationbuffer over a MicroCon filter (10 kDa cut off) to remove excess peptide.The successful coupling was detected by an altered running performancein an SDS polyacrylamide gel electrophoresis. FIG. 11 shows twoindependently generated preparations of H6UvsX*(RGD2) on an SDSpolyacrylamide gel. The coupled peptide results in an increase of themolecular weight and thus in an alteration of the running performance inSDS gels.

Description of the Experiment:

Binding of UvsX-NLS and UvsX-RGD to double-stranded DNA and formation ofmixed NPFs:

Reactions with 140 ng (in each case) of a purified 1.6 kb PCR DNAfragment with a mixture of 15 μg H6UvsX*(RGD2) and 4 μg UvsXH6N2-2, with4 μg UvsXH6N2-2 alone and with 15 μg H6UvsX*(RGD2) alone were incubatedin 76 mM K₂HPO₄, 17 mM KH₂PO₄, 14 mM NaH₂PO₄, pH=7.2, 5 mM MgCl₂ and 1mM ATP-γ-S in a final volume of 20 μl for 30 min at room temperature andwere then transferred to a 0.8% TAE/agarose gel which was afterwardsstained with ethidium bromide. The electrophoresis was carried out at100 V for 1 h.

FIG. 12 shows that the two differently modified proteins form NPFs withthe DNA which markedly differ in their running performance. NPFsconsisting of a double-stranded DNA fragment and a mixture ofH6UvsX*(RGD2) and UvsXH6N2-2 (lane 1), or of UvsXH6N2-2 alone (lane 2)or of H6UvsX*(RGD2) alone (lane 3), were separated electrophoreticallyin an agarose gel. Since protein is present in low amounts, free DNAfragment is present as well. Both proteins in a reaction bind togetherto the DNA fragment and result in a mixed NPF that has a molecularweight that lies between that of the NPFs of the pure proteins (lane 1).From this it can be concluded that UvsX-NLS and UvsX-RGD bind todouble-stranded DNA and can form mixed NPFs.

Example 10 Specific Uptake of NPFs into Cells by Integrin-MediatedEndocytosis

Structure of the Proteins

As in example 1, plasmid UvsXH6N2NIT-2 (FIG. 1) which permits theexpression of a fusion protein of UvsX and an integrin-binding RGDmotif, was generated recombinantly.

Amino acids 1-391: UvsX from the phage T4 (NCBI protein accession no.:AAD42669, amino acids 1-391), amino acids 392-394: linker consisting ofthe amino acids S³⁹²YG³⁹⁴, amino acids 395-400: H³⁹⁵ HHHHH⁴⁰⁰, aminoacids 401-403: linker consisting of the amino acids M⁴⁰¹YS⁴⁰³, aminoacids 404-417: nuclear localization signal nls-2 (amino acids 2-15, SEQID NO: 9 from WO 00/40742), amino acids 418-420: C-terminus consistingof the amino acids G⁴¹⁸YP⁴²⁰ and amino acids 421-432: RGD motif “NIT”:N⁴²¹TRGDTYIPYP⁴³² (SEQ ID NO:5).

Description of the Experiment:

Generation of NPFs from fluorescence-labeled DNA and UvsX derivatives:

1 μg each of a purified 1.6 kb PCR DNA fragment containingAlexaFluor488-labeled dUTP (Molecular Probes, Eugene, Oreg., USA)instead of dTTP, was incubated in 76 mM K₂HPO₄, 17 mM KH₂PO₄, 14 mMNaH₂PO₄, pH=7.2, 5 mM MgCl₂ and 1 mM ATP-γ-S with 100 μg purifiedUvsXH6N2 or UvsXH6N2NIT-2 in a final volume of 200 μl for 30 min at roomtemperature. Then, 10 μl of each NPF reaction was applied to a 0.8%TAE/agarose gel which was subsequently stained with ethidium bromide.The electrophoresis was carried out for 1 hour at 100 volts. FIG. 13shows that the DNA was completely retarded. Thus, the DNA binding of theproteins is not hampered by the fluorescein labelling of the DNA.

Uptake of the NPFs in NIH3T3 cells by endocytosis:

NIH3T3 cells were plated in 6 well plates (3×10⁵ per well), incubatedover night at 37° C. and 5% CO₂, and were washed the next morning withprewarmed FCS-free medium; afterwards 2 ml FCS-free medium was added.190 μl of the NPF reactions were added (see above), incubated for 30 minat room temperature, the supernatant was removed, the cells were washedand covered with 3 ml medium (with 10% FCS). After another incubationfor 1 hour at 37° C. in the incubator, the analysis was carried outunder the fluorescence microscope. In FIGS. 14 a and 14 b, one pictureeach is shown in bright field (lower) and reflected light fluorescence(upper)

In the bright field, vesicular intracellular compartments are visiblewhich glow in the fluorescent light due to the endocytosed DNA (FIG. 14a, b, upper).

From each well, several pictures were taken. The cells were counted andthe proportion of cells containing at least one fluorescent vesicularcompartment was determined in percent (shown in FIG. 15). Each pictureshows a mean of 35 cells. Of the reactions with UvsXH6N2NIT-2, ninepictures were analyzed, and of the reactions with UvsXH6N2-2, fivepictures were analyzed.

The result shows that the modification of UvsX with an integrin-bindingmotif as a functional component (UvsXH6N2-NIT-2) results in a markedlyincreased endocytotic uptake of the transfection agents in the cellscompared to the control (UvsXH6N2-2).

Example 11 Generation of a Transfection Agent Based on hRad51 asNPF-Forming Protein

The proteins hRad51H6 and hRad51H6N2 were used as NPF-forming proteins(see FIG. 16).

Structure of the Proteins

hRad51H6 (352 amino acids):

Amino acids 1-339: human Rad51 (NCBI protein accession no: Q06609, aminoacids 1-339), amino acids 340-343: linker consisting of the amino acidsY³⁴⁰SYG³⁴³, amino acids 344-349: H³⁴⁴HHHHH³⁴⁹ for purification by nickelchelate affinity chromatography, amino acids 350-352: C-terminusconsisting of the amino acids M³⁵⁰YS³⁵².

hRad51H6N2 (369 amino acids):

Amino acids 1-339: human Rad51 (NCBI protein accession no: Q06609, aminoacids 1-339), amino acids 340-343: linker consisting of the amino acidsY³⁴⁰ SYG³⁴³, amino acids 344-349: H³⁴⁴HHHHH³⁴⁹ for purification bynickel chelate affinity chromatography, amino acids 350-352: linkerconsisting of the amino acids M³⁵⁰YS³⁵², amino acids 353-366: nuclearlocalization signal nls-2 (amino acids 2-15, SEQ ID NO: 9 from WO00/40742), amino acids 367-369: C-terminus consisting of the amino acidsG³⁶⁷YP³⁶⁹.

Cloning of the Expression Plasmids

For expression of the above mentioned proteins in suitable Escherichiacoli cells, plasmids were constructed that contain a coding sequence forhRad51H6 or hRad51H6N2 under the control of the lac promoter(pExH-hRad51H6 or pExH-hRad51H6N2, see FIG. 16). pExH-UvsXH6-2 andpExH-UvsXH6N2-2 were used as source plasmids (see FIG. 16). The codingregion for UvsX was cut out with EcoR V and BsiW I, and replaced by aPCR fragment with the coding region for hRad51 which had been cut out inthe same way. This was amplified from a human cDNA library usinghRad51-specific primers that contain the desired restriction sites. Atthe 5′-end before the start codon, the PCR products contained theadditional nucleotides 5′-CACACATCTAGACGTACGGATATCAT-3′ (SEQ ID NO:6),and at their 3′-end they contained the additional nucleotides5′-TACTCGTACGGAGGTGGCGGCCGCTGTGTG-3′ (SEQ ID NO:7) instead of the stopcodon.

Purification:

A preculture of 5 ml dYT/ampicillin (100 μg/ml) was inoculated with acolony of pExH-Rad51H6 or pExH-Rad51H6N2 in DH5, and was grown for 5hours at 37° C. with 250 rpm. 10 l dYT/ampicillin (100 μg/ml) wasinoculated with this preculture, and was allowed to grow for another 24hours at 37° C. with 210 UPM. The cultures were harvested at 7000×g, andyielded approx. 30-50 g bacterial pellet. This was frozen for 1-3 daysat −20° C. The pellet was thawed on ice, and resuspended in 100 ml coldstarting buffer. The cells were then solubilized by ultrasound using aB. Braun Labsonic U (large probe, parameters: 300 watts, 0.5 sec pulseduration per second, 8 min sonification). Then it was incubated with 10mg lysozyme (Serva, 190.000 u/mg) for 1 hours at 4° C. and for another30 min after addition of 50 μl DNAse I (Serva, 2 mg/ml) with slowstirring. After removing the lysate by centrifugation (45 min, 18000×g,4° C.), the supernatant was filtered through sterile filters (pore sizes0.45 μm and 0.2 μm) and loaded on an equilibrated 1 ml HiTrap™ chelatingcolumn (Pharmacia) preloaded with Ni⁺⁺ ions. The further purificationsteps were carried out according to the respective Pharmacia protocolfor proteins that have been provided with a histidine hexamer. Aliquotsof the various elution fractions were applied to SDS/Coomassie gels. Thepurest fractions were combined and were further concentrated overCentriplus YM30 columns (Millipore) according to the respectiveprotocol. Then it was dialyzed twice (dialysis tubing: Spectra/Por,MWCO: 25.000) against an at least one thousand-fold volume of ZI bufferfor 1 hour each at 4° C., then over night at 4° C. against ZI buffer/50%glycerine. The dialysis product was aliquoted in 30-50 μl fractions andstored at −80° C. FIG. 17A shows the purified hRad51-H6 and hRad51-H6N2proteins.

Used Buffer:

As in example 1, but different elution buffer: 20 mM Pi, 0.5 M NaCl,

100-1000 mM imidazole, pH=7.4

Determination of Concentration:

The concentrations of the hRad51 proteins was determined by measurementof the OD₂₈₀ using the extinction coefficient calculated with the GeneInspector™ software (Textco, Inc.).

With hRad51H6 and hRad51H6N2 it was 11-13 μg/μl.

Description of the Experiment:

Binding of NLS-modified hRad51 to double-stranded DNA:

hRad51H6N2 purified over Ni⁺⁺-sepharose was incubated with 100 ng eachof a 0.9 kb PCR fragment.

A DNA shift in the agarose gel caused by the protein binding shows thathRad51H6N2 cooperatively binds double-stranded DNA based on theconcentration (FIG. 17B). Even with low amounts of hRad51H6N2, singleDNA molecules are completely bound by hRad51H6N2, and are thereforeretarded maximally, so that the retardation of the DNA does not increaseany more by increasing the amount of protein. It is concluded thathRad51H6N2 binds dsDNA.

Example 12 Generation of a Transfection Agent Based on UvsX with aSignal for the Non-Endosomal Membrane Permeation and a NuclearLocalization Signal as Functional Component

As in example 1, a plasmid was generated that permits the expression ofthe fusion protein UvsXH6N2VP22c50 (see FIG. 1), which additionallycontains a part of the tegument protein VP22 (gene UL49) of the humanherpes virus 1 and is based on the protein UvsXH6N2-2 described inexample 2. The here used VP22 peptide acts as a signal for thenon-endosomal permeation through the cell membrane. Thus, the fusionprotein UvsXH6N2VP22c50 contains a membrane transduction signal inaddition to a nuclear localization signal (NSL).

Structure of the Protein

UvsXH6N2VP22c50 (474 amino acids):

Amino acids 1-391: UvsX from the phage T4 (NCBI protein accession no:AAD42669, amino acids 1-391), amino acids 392-394: linker consisting ofthe amino acids S³⁹² YG³⁹⁴, amino acids 395-400: H³⁹⁵HHHHH⁴⁰⁰, aminoacids 401-403: linker consisting of the amino acids M⁴⁰¹YS⁴⁰³, aminoacids 404-417: nuclear localization signal nls-2 (amino acids 2-15, SEQID NO: 9 aus WO 00/40742), amino acids 418-422: linker consisting of theamino acids G⁴¹⁸YPGS⁴²², amino acids 423-472: part of the tegumentprotein VP22 (Gen UL49) of the human herpes virus 1 (NCBI proteinaccession no: NP_(—)044651, amino acids 252-301), amino acids 473-474:C-terminus consisting of the amino acids P⁴⁷³R⁴⁷⁴.

Cloning of the Expression Plasmid:

For the expression in suitable Escherichia coli cells, pExHUvsXH6N2-2(see example 1 and FIG. 1) was opened by restriction enzyme digestionwith Acc65 I and Spe I, and was ligated with a PCT product cut withAcc65 I and Nhe I, which contains at the 5′-end the additionalnucleotides 5′-CACACAGGTACCCGGGATCC-3′ (SEQ ID NO:8) and at its 3′-endthe additional nucleotides 5′-CCTAGGTAATAATAAGCGGCCGCGCTAGCTGTGTG-3′(SEQ ID NO:9), in addition to the coding sequence for the last 50 aminoacids of the tegument protein VP22 (gene UL49) of the human herpes virus1 (NCBI nucleotide accession no: NC_(—)001806, complementary sequence ofthe nucleotides 105486-106391) (see FIG. 1).

Purification:

The purification of UvsXH6N2VP22c50 was carried out as described inexample 1 for H6UvsX (see FIG. 18A).

Concentration: 1.8 μg/11.

Description of the Experiment:

Binding of a mixture of various modified UvsX (UvsX-NLS-VP22 andUvsX-NLS) to double-stranded DNA:

140 ng (in each case) of a purified 1.7 kb PCR fragments were incubatedin 96 mM K₂HPO₄, 21.5 mM KH₂PO₄, 18 mM NaH₂PO₄, pH=7.2, 5 mM MgCl₂ and1.3 mM ATP-Y-S with the amounts according to FIG. 18B of purifiedUvsXH6N2VP22c50 or UvsXH6N2-2 for 30 min at room temperature, then, allreactions were applied to a 0.8% TAE/agarose gel which was afterwardsstained with ethidium bromide. The two proteins were different regardingtheir molecular weight and net charge, and therefore retarded the DNAdifferently during electrophoresis, with the complex withUvsXH6N2VP22c50 remaining stuck in the gel pocket and not migrating anymore (see lanes 1 and 7 of FIG. 18B). When the proteins are mixed beforethey are added to the DNA, intermediate complexes are formed dependingon the ratio of UvsXH6N2-2 and UvsXH6N2VP22c50, the migrationperformance of which is between those of the unmixed complexes (FIG.18B). This shows that the DNA is occupied by both proteins. Alsopossible is a mixture of differently modified or one- or two-timesmodified NPF-forming proteins.

Example 13 Transfection of a Cell Line (NIH3T3) with Complexes of DNAand a Mixture of UvsX-NLS-VP22 and UvsX-NLS

Description of the Experiment:

2.5×10⁵ cells (NIH3T3) were plated out in each well of a 6-well plate,and transfected on the following day with a vector containing a gene forthe expression of a fluorescent reporter protein. For this, 0 μg-1 μglinear or 1 μg circular DNA was preincubated with 36 μg UvsXH6N2VP22c50or a mixture of 19 μg UvsXH6N2VP22c50 and 39 μg UvsXH6N2-2 in bindingbuffer (76 mM K₂HPO₄, 17 mM KH₂PO₄, 14 mM NaH₂PO₄, 5 mM MgCl₂, 1 mMATP-γ-S, pH 7.21) for 30 min at room temperature, and together with 1 mlRPMI was added to cells that have before been washed once with PBS/BSA.After a 1 h incubation at 37° C., 5% CO₂ in the incubator, 1 ml RPMI/20%FCS was added respectively and further incubated in the incubator. 4 hor 24 h later, the cells were analyzed in the fluorescence microscope.Cells were observed that expressed the reporter gene after treatmentwith complexes of linear or circular DNA and the mixture ofUvsXH6N2VP22c50 and UvsXH6N2-2 (see FIG. 19). The number of thetransfected cells increased with the amount of used DNA or DNA-proteincomplexes.

However, no expression of the reporter gene was achieved with DNAcomplexes containing only UvsXH6N2VP22c50, or in absence of DNA.

Therefore it appears that by modification of an NPF-forming protein,here UvsX, with a membrane-active peptide, here VP22, the transfectionof cells and here especially the membrane permeation is facilitated. Themodular character of the method or transfection agent according to theinvention is underlined by the combination of differently modifiedproteins, here modification with VP22 and NLS (see also FIG. 20).Whereas the VP22-modified UvsX permits the non-endosomal membranepermeation, the NLS-modified UvsX directs the transfected DNA from thecytoplasma to the nucleus; the transfected DNA can then be expressedthere.

Thus, individual steps of the complex transfection procedure can becontrolled specifically, flexible and with high efficiency in especiallyadvantageous manner.

REFERENCES

-   Andersson, K. (1990). Codon Preferences in Free-Living    Microorganisma. Micobiol Rev 54, 98-210.-   Bal, H. P., Chroboczek, J., Schoehn, G., Ruigrok, R. W., and    Dewhurst, S. (2000). Adenovirus type 7 penton purification of    soluble pentamers from Escherichia coli and development of an    integrin-dependent gene delivery system. Eur J Biochem 267,    6074-6081.-   Baumann, P., and West, S. C. (1998). Role of the human RAD51 protein    in homologous recombination and double-stranded-break repair. Trends    Biochem Sci 23, 247-251.-   Bianco, P. R., Tracy, R. B., and Kowalczykowski, S. C. (1998). DNA    strand exchange proteins: a biochemical and physical comparison.    Front Biosci 3, D570-603.-   Boulikas, T. (1993). Nuclear localization signals (NLS). Crit. Rev    Eukaryot Gene Expr 3, 193-227.-   Boulikas, T. (1996). Nuclear import of protein kinases and cyclins.    J Cell Biochem 60, 61-82.-   Boulikas, T. (1997). Nuclear import of DNA repair proteins.    Anticancer Res 17, 843-863.-   Cagnon, C., Valverde, V., and Masson, J. M. (1991). A new family of    sugar-inducible expression vectors for Escherichia coli. Protein Eng    4, 843-847.-   Cerutti, H., Osman, M., Grandoni, P., and Jagendorf, A. T. (1992). A    homolog of Escherichia coli RecA protein in plastids of higher    plants. Proc Natl Acad Sci USA 89, 8068-8072.-   Collins, L., Sawyer, G. J., Zhang, X. H., Gustafsson, K., and    Fabre, J. W. (2000). In vitro investigation of factors important for    the delivery of an integrin-targeted nonviral DNA vector in organ    transplantation. Transplantation 69, 1168-1176.-   Delcayre, A. X., Salas, F., Mathur, S., Kovats, K., Lotz, M., and    Lernhardt, W. (1991). Epstein Barr virus/complement C3d receptor is    an interferon alpha receptor. Embo J 10, 919-926.-   Di Capua, E., Engel, A., Stasiak, A., and Koller, T. (1982).    Characterization of complexes between recA protein and duplex DNA by    electron microscopy. J Mol Biol 157, 87-103.-   Ellouze, C., Selmane, T., Kim, H. K., Tuite, E., Norden, B.,    Mortensen, K., and Takahashi, M. (1999). Difference between active    and inactive nucleotide cofactors in the effect on the DNA binding    and the helical structure of RecA filament dissociation of RecA-DNA    complex by inactive nucleotides. Eur J Biochem 262, 88-94.-   Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985).    Isolation of monoclonal antibodies specific for human c-myc    proto-oncogene product. Mol Cell Biol 5, 3610-3616.-   Feero, W. G., Li, S., Rosenblatt, J. D., Sirianni, N., Morgan, J.    E., Partridge, T. A., Huang, L., and Hoffman, E. P. (1997).    Selection and use of ligands for receptor-mediated gene delivery to    myogenic cells. Gene Ther 4, 664-674.-   Feldherr, C. M., and Akin, D. (1997). The location of the transport    gate in the nuclear pore complex. J Cell Sci 110 (Pt 24), 3065-3070.-   Fominaya, J., and Wels, W. (1996). Target cell-specific DNA transfer    mediated by a chimeric multidomain protein. Novel non-viral gene    delivery system. J Biol Chem 271, 10560-10568.-   Griffith, J., Makhov, A., Santiago-Lara, L., and Setlow, P. (1994).    Electron microscopic studies of the interaction between a Bacillus    subtilis alpha/beta-type small, acid-soluble spore protein with DNA:    protein binding is cooperative, stiffens the DNA, and induces    negative supercoiling. Proc Natl Acad Sci USA 91, 8224-8228.-   Harbottle, R. P., Cooper, R. G., Hart, S. L., Ladhoff, A., McKay,    T., Knight, A. M., Wagner, E., Miller, A. D., and Coutelle, C.    (1998). An RGD-oligolysine peptide: a prototype construct for    integrin-mediated gene delivery. Hum Gene Ther 9, 1037-1047.-   Hong, S. S., Gay, B., Karayan, L., Dabauvalle, M. C., and    Boulanger, P. (1999). Cellular uptake and nuclear delivery of    recombinant adenovirus penton base. Virology 262, 163-177.-   Karlin, S., and Brocchieri, L. (1996). Evolutionary conservation of    RecA genes in relation to protein structure and function. J    Bacteriol 178, 1881-1894.-   Karlin, S., Weinstock, G. M., and Brendel, V. (1995). Bacterial    classifications derived from recA protein sequence comparisons. J    Bacteriol 177, 6881-6893.-   Knight, K. L., and McEntee, K. (1985). Affinity labeling of a    tyrosine residue in the ATP binding site of the recA protein from    Escherichia coli with 5′-p-fluorosulfonylbenzoyladenosine. J Biol    Chem 260, 10177-10184.-   Kukowska-Latallo, J. F., Bielinska, A. U., Johnson, J., Spindler,    R., Tomalia, D. A., and Baker, J. R., Jr. (1996). Efficient transfer    of genetic material into mammalian cells using Starburst    polyamidoamine dendrimers. Proc Natl Acad Sci USA 93, 4897-4902.-   Lee, C. K., and Knipe, D. M. (1985). An immunoassay for the study of    DNA-binding activities of herpes simplex virus protein ICP8. J Virol    54, 731-738.-   Masson, J. Y., Davies, A. A., Hajibagheri, N., Van Dyck, E.,    Benson, F. E., Stasiak, A. Z., Stasiak, A., and West, S. C. (1999).    The meiosis-specific recombinase hDmc1 forms ring structures and    interacts with hRad51. Embo J 18, 6552-6560.-   Mengaud, J., Ohayon, H., Gounon, P., Mege, R. M., and Cossart, P.    (1996). E-cadherin is the receptor for internalin, a surface protein    required for entry of L. monocytogenes into epithelial cells. Cell    84, 923-932.-   Midoux, P., Mendes, C., Legrand, A., Raimond, J., Mayer, R.,    Monsigny, M., and Roche, A. C. (1993). Specific gene transfer    mediated by lactosylated poly-L-lysine into hepatoma cells. Nucleic    Acids Res 21, 871-878.-   Mosig, G. (1987). The essential role of recombination in phage T4    growth. Annu Rev Genet. 21, 347-371.-   Neumann, G., Castrucci, M. R., and Kawaoka, Y. (1997). Nuclear    import and export of influenza virus nucleoprotein. J Virol 71,    9690-9700.-   Ogawa, T., Shinohara, A., Nabetani, A., Ikeya, T., Yu, X.,    Egelman, E. H., and-   Ogawa, H. (1993). RecA-like recombination proteins in eukaryotes:    functions and structures of RAD51 genes. Cold Spring Harb Symp Quant    Biol 58, 567-576.-   Ohno, K., Sawai, K., lijima, Y., Levin, B., and Meruelo, D. (1997).    Cell-specific targeting of Sindbis virus vectors displaying    IgG-binding domains of protein A. Nat Biotechnol 15, 763-767.-   Pack, D. W., Putnam, D., and Langer, R. (2000). Design of    imidazole-containing endosomolytic biopolymers for gene delivery.    Biotechnol Bioeng 67, 217-223.-   Plow, E. F., Haas, T. A., Zhang, L., Loftus, J., and Smith, J. W.    (2000). Ligand binding to integrins. J Biol Chem 275, 21785-21788.-   Pooga, M., Soomets, U., Hallbrink, M., Valkna, A., Saar, K., Rezaei,    K., Kahl, U., Hao, J. X., Xu, X. J., Wiesenfeld-Hallin, Z., et al.    (1998). Cell penetrating PNA constructs regulate galanin receptor    levels and modify pain transmission in vivo. Nat Biotechnol 16,    857-861.-   Provoda, C. J., and Lee, K. D. (2000). Bacterial pore-forming    hemolysins and their use in the cytosolic delivery of    macromolecules. Adv Drug Deliv Rev 41, 209-221.-   Richardson, S., Ferruti, P., and Duncan, R. (1999).    Poly(amidoamine)s as potential endosomolytic polymers: evaluation in    vitro and body distribution in normal and tumour-bearing animals. J    Drug Target 6, 391-404.-   Roca, A. I., and Cox, M. M. (1990). The RecA protein: structure and    function. Crit. Rev Biochem Mol Biol 25, 415-456.-   Rosenkranz, A. A., Yachmenev, S. V., Jans, D. A., Serebryakova, N.    V., Murav'ev, V. I., Peters, R., and Sobolev, A. S. (1992).    Receptor-mediated endocytosis and nuclear transport of a    transfecting DNA construct. Exp Cell Res 199, 323-329.-   Sandler, S. J., Satin, L. H., Samra, H. S., and Clark, A. J. (1996).    recA-like genes from three archaean species with putative protein    products similar to Rad51 and Dmc1 proteins of the yeast    Saccharomyces cerevisiae. Nucleic Acids Res 24, 2125-2132.-   Schägger, H., and von Jagow, G. (1987). Tricine-Sodium Dodecyl    Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of    Proteins in the Range from 1 to 100 kDa. Anal Biochem 166, 368-379.-   Schoeman, R., Joubert, D., Ariatti, M., and Hawtrey, A. O. (1995).    Further studies on targeted DNA transfer to cells using a highly    efficient delivery system of biotinylated transferrin and    biotinylated polylysine complexed to streptavidin. J Drug Target 2,    509-516.-   Seitz, E. M., Brockman, J. P., Sandler, S. J., Clark, A. J., and    Kowalczykowski, S. C. (1998). RadA protein is an archaeal RecA    protein homolog that catalyzes DNA strand exchange. Genes Dev 12,    1248-1253.-   Steinhauer, D. A., Wharton, S. A., Skehel, J. J., and Wiley, D. C.    (1995). Studies of the membrane fusion activities of fusion peptide    mutants of influenza virus hemagglutinin. J Virol 69, 6643-6651.-   Surdej, P., and Jacobs-Lorena, M. (1994). Strategy for epitope    tagging the protein-coding region of any gene. Biotechniques 17,    560-565.-   Tang, M. X., and Szoka, F. C. (1997). The influence of polymer    structure on the interactions of cationic polymers with DNA and    morphology of the resulting complexes. Gene Ther 4, 823-832.-   Thoren, P. E., Persson, D., Karlsson, M., and Norden, B. (2000). The    antennapedia peptide penetratin translocates across lipid    bilayers—the first direct observation. FEBS Lett 482, 265-268.-   Thyagarajan, B., Padua, R. A., and Campbell, C. (1996). Mammalian    mitochondria possess homologous DNA recombination activity. J Biol    Chem 271, 27536-27543.-   Wagner, E. (1999). Application of membrane-active peptides for    nonviral gene delivery. Adv Drug Deliv Rev 38, 279-289.-   Wang, P., Palese, P., and O'Neill, R. E. (1997). The NPI-1/NPI-3    (karyopherin alpha) binding site on the influenza a virus    nucleoprotein NP is a nonconventional nuclear localization signal. J    Virol 71, 1850-1856.-   Weisbart, R. H., Baldwin, R., Huh, B., Zack, D. J., and    Nishimura, R. (2000). Novel protein transfection of primary rat    cortical neurons using an antibody that penetrates living cells. J    Immunol 164, 6020-6026.-   Yamada, M., and Kasamatsu, H. (1993). Role of nuclear pore complex    in simian virus 40 nuclear targeting. J Virol 67, 119-130.-   Yu, X., and Egelman, E. H. (1993). DNA conformation induced by the    bacteriophage T4 UvsX protein appears identical to the conformation    induced by the Escherichia coli RecA protein. J Mol Biol 232, 1-4.-   Zauner, W., Blaas, D., Kuechler, E., and Wagner, E. (1995).    Rhinovirus-mediated endosomal release of transfection complexes. J    Virol 69, 1085-1092.

1. A transfection agent for transfecting cells with a nucleic acid,comprising at least one protein being capable of forming nucleoproteinfilaments (NPFs), said nucleic acid and said NPF-forming protein forminga nucleoprotein filament complex, wherein said NPF-forming protein is aprotein selected from the group consisting of the proteins RadA, hDmc1,SASP, ICP8, and UvsX, or a mixture of at least 2 of the listed proteins,wherein said NPF-forming protein is modified by at least onemodification selected from the group consisting of deletion or insertionof amino acids and/or protein domains, chemical alteration of aminoacids, and chemical coupling of peptides, proteins, carbohydrates orlipids, wherein said NPF-forming protein is modified with a singlefunctional component or several functional components of the samefunction, wherein said functional component(s) cause(s) association ofsaid complex to the cellular surface, non-endosomal passage of saidcomplex through the cell membrane, release of said complex fromendosomes/lysosomes, or transport of said complex into the nucleus, andwherein said functional component(s) is/(are) selected from the groupconsisting of amino acid linker, Nuclear Localization Signal (NLS),ligand to the Epstein Barr Virus receptor CD21, ligand to Listeriamonocytogenes receptor E-cadherin, ligand to transferrinreceptor/transferrin, ligand to asialoglycoprotein receptor,integrin-binding peptides, ligand to insulin receptor, ligand to EGF(epidermal growth factor) receptor, ligand to insulin-like growth factorI receptor, ligand to lectins, protein A or its IgG-binding domain,epitope from the influenza hemagglutinin, epitope from the c-mycprotein, viral peptides including peptides from HIV tat, peptides fromVP22, peptides from HBV surface antigen, peptides from homeodomain ofantennapedia, peptides from engrailed, peptides from HOXA-5, peptidesfrom IL-1β, peptides from FGF-1, peptides from FGF-2, peptides fromKaposi fibroblast growth factor, mab 3E10, transportane, endosomolyticsubstances including endosomolytic peptides from bacteria includingpeptides from streptolysin O, peptides from pneumolysin, peptides fromstaphylococcal α-toxin, peptides from listeriolysin O, endosomolyticpeptides from viruses including peptides from the N-terminalhemagglutinin HA-2 peptide of influenza virus, peptides from theN-terminus of the VP-1 protein of rhinovirus HRV2, peptides from thecapsid component Ad2 of adenovirus, synthetic endosomolytic peptidesincluding amphipathic peptide GALA, amphipathic peptide KALA,amphipathic peptide EGLA, amphipathic peptide JTS1, imidazole, andpolyamidoamine-modified polymers, or a mixture of at least 2 of thelisted modifications.
 2. The transfection agent according to claim 1,wherein different NPF-forming proteins are each modified with componentsof the same function.
 3. The transfection agent according to claim 1,wherein an integrin/integrin-binding peptide is coupled to saidNPF-forming protein.
 4. The transfection agent according to claim 3,wherein said integrin/integrin-binding peptide is RGD.
 5. Thetransfection agent according to claim 1, wherein a viral peptide iscoupled to said NPF-forming protein.
 6. The transfection agent accordingto claim 5, wherein said viral peptide is VP22.
 7. The transfectionagent according to claim 1, wherein an endosomolytic substance iscoupled to said NPF-forming protein.
 8. The transfection agent accordingto claim 7, wherein said endosomolytic substance is Streptolysin O. 9.The transfection agent according to claim 8, wherein a nuclearlocalization signal (NLS) is coupled to said NPF-forming protein. 10.The transfection agent according to claim 9, wherein said nuclearlocalization signal is the NLS of the large T-antigen of SV40.
 11. Thetransfection agent according to claim 1, wherein VP22 is coupled to saidNPF-forming protein.
 12. The transfection agent according to claim 1,wherein a nuclear localization signal is coupled to said NPF-formingprotein.
 13. The transfection agent according to claim 1, wherein saidNPF-forming protein is selected from the group consisting of theproteins SASP and ICP8, and wherein RGD, VP22, a NLS or an endosomolyticsubstance is coupled to said NPF-forming protein.
 14. A kit, suitablefor transfecting cells with a nucleic acid, comprising at least oneprotein being capable of forming nucleoprotein filaments (NPFs), saidnucleic acid and said NPF-forming protein forming a nucleoproteinfilament complex, wherein said NPF-forming protein is a protein selectedfrom the group consisting of the proteins RadA, hDmc1, SASP, ICP8, andUvsX, or a mixture of at least 2 of the listed proteins, and furthercomprising at least one of the following components: nucleosidetriphosphate and/or nucleoside triphosphate analogues, at least onenucleic acid to be transfected, adjuvants and additives, wherein saidNPF-forming protein is modified by at least one modification selectedfrom the group consisting of deletion or insertion of amino acids and/orprotein domains, chemical alteration of amino acids, and chemicalcoupling of peptides, proteins, carbohydrates or lipids, wherein saidNPF-forming protein is modified with a single functional component orseveral functional components of the same function, wherein saidfunctional component(s) cause(s) association of said complex to thecellular surface, non-endosomal passage of said complex through the cellmembrane, release of said complex from endosomes/lysosomes, or transportof said complex into the nucleus, and wherein said functionalcomponent(s) is/(are) selected from the group consisting of amino acidlinker, Nuclear Localization Signal (NLS), ligand to the Epstein BarrVirus receptor CD21, ligand to Listeria monocytogenes receptorE-cadherin, ligand to transferrin receptor/transferrin, ligand toasialoglycoprotein receptor, integrin-binding peptides, ligand toinsulin receptor, ligand to EGF (epidermal growth factor) receptor,ligand to insulin-like growth factor I receptor, ligand to lectins,protein A or its IgG-binding domain, epitope from the influenzahemagglutinin, epitope from the c-myc protein, peptides from HIV tat,peptides from VP22, peptides from HBV surface antigen, peptides fromhomeodomain of antennapedia, peptides from engrailed, peptides fromHOXA-5, peptides from IL-1β, peptides from FGF-1, peptides from FGF-2,peptides from Kaposi fibroblast growth factor, mab 3E10, transportane,peptides from streptolysin O, peptides from pneumolysin, peptides fromstaphylococcal α-toxin, peptides from listeriolysin O, peptides from theN-terminal hemagglutinin HA-2 peptide of influenza virus, peptides fromthe N-terminus of the VP-1 protein of rhinovirus HRV2, peptides from thecapsid component Ad2 of adenovirus, amphipathic peptide GALA,amphipathic peptide KALA, amphipathic peptide EGLA, amphipathic peptideJTS1, imidazole, and polyamidoamine-modified polymers, or a mixture ofat least 2 of the listed modifications.
 15. A method for transfectingcells using at least one protein being capable of forming nucleoproteinfilaments, said method comprising: providing at least one NPF-formingprotein selected from the group consisting of the proteins RadA, hDmc1,SASP, ICP8, and UvsX, or a mixture of at least 2 of the listed proteins,modifying said protein by providing at least one modification selectedfrom the group consisting of deletion or insertion of amino acids and/orprotein domains, chemical alteration of amino acids, and chemicalcoupling of peptides, proteins, carbohydrates or lipids, wherein saidNPF-forming protein consists of a NPF-forming protein that is modifiedwith a single functional component or several functional components ofthe same function, wherein said function is selected from the groupconsisting of association of said complex to the cellular surface,non-endosomal passage of said complex through the cell membrane, releaseof said complex from endosomes/lysosomes, and transport of said complexinto the nucleus, and wherein said functional component(s) is/(are)selected from the group consisting of amino acid linker, NuclearLocalization Signal (NLS), ligand to the Epstein Barr Virus receptorCD21, ligand to Listeria monocytogenes receptor E-cadherin, ligand totransferrin receptor/transferrin, ligand to asialoglycoprotein receptor,integrin-binding peptides, ligand to insulin receptor, ligand to EGF(epidermal growth factor) receptor, ligand to insulin-like growth factorI receptor, ligand to lectins, protein A or its IgG-binding domain,epitope from the influenza hemagglutinin, epitope from the c-mycprotein, peptides from HIV tat, peptides from VP22, peptides from HBVsurface antigen, peptides from homeodomain of antennapedia, peptidesfrom engrailed, peptides from HOXA-5, peptides from IL-1β, peptides fromFGF-1, peptides from FGF-2, peptides from Kaposi fibroblast growthfactor, mab 3E10, transportane, peptides from streptolysin O, peptidesfrom pneumolysin, peptides from staphylococcal α-toxin, peptides fromlisteriolysin O, peptides from the N-terminal hemagglutinin HA-2 peptideof influenza virus, peptides from the N-terminus of the VP-1 protein ofrhinovirus HRV2, peptides from the capsid component Ad2 of adenovirus,amphipathic peptide GALA, amphipathic peptide KALA, amphipathic peptideEGLA, amphipathic peptide JTS1, imidazole, polyamidoamine-modifiedpolymers, and a mixture of at least 2 of the said functional components;loading a nucleic acid to be transfected with the modified protein, thenucleic acid and the modified protein forming a nucleoprotein filamentcomplex; and adding said complex to the cells to be transfected, whereinsaid modification causes association of said complex to the cellularsurface, non-endosomal passage of said complex through the cellmembrane, release of said complex from endosomes/lysosomes, or transportof said complex into the nucleus.
 16. The method according to claim 15,wherein said complex is stabilized by addition of nucleosidetriphosphates and/or non-hydrolyzable analogues thereof.
 17. The methodaccording to claim 15 being employed in combination with otherbiological and/or chemical and/or physical transfection methods fornucleic acids.
 18. The method according to claim 16, wherein thenucleoside triphosphates and/or non-hydrolyzable analogues thereof areATP (adenosintriphosphate) and/or GTP (guanosintriphosphate) and/ortheir non-hydrolyzable analogues.
 19. The method according to claim 17,wherein the biological and/or chemical and/or physical transfectionmethods for nucleic acids are electroporation.